Mendelian susceptibility to mycobacterial disease (MSMD) is a primary immunodeficiency syndrome characterized by severe disease caused by weakly virulent mycobacteria, such as BCG vaccines and environmental mycobacteria, in otherwise healthy patients. Patients with MSMD are also vulnerable to tuberculosis. Other infections are rare, with the exception of non-typhoidal salmonellosis, found in about half the patients. First described clinically in the 1950s, the pathogenesis of MSMD remained unclear until 1996, when its first genetic etiology was deciphered in children with interferon-3 receptor 1 (IFN-?R1) deficiency. Genetic dissection of MSMD over the last 15 years has identified six morbid genes, including five autosomal (IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1) and one X-linked (NEMO) gene. The high level of allelic heterogeneity at these six loci has led to the definition of up to 13 distinct disorders, with dominant and recessive traits at the same locus, complete and partial defects of the same protein, and defects due to a lack of protein or the production of a dysfunctional protein. The pathogenesis of MSMD in patients with these disorders involves impaired interleukin-12 (IL-12)-dependent IFN-? immunity. However, only about half the 500 patients tested in our laboratory carried any of these genetic defects. We hypothesize that MSMD in other patients results from other monogenic inborn errors of immunity, possibly but not necessarily involving the IL- 12-IFN-? circuit. The principal objective of the work described in this application is therefore to identify new MSMD-causing genetic disorders, including new defects allelic with the known MSMD etiologies, and new MSMD-causing genes. We will use two complementary experimental approaches, a hypothesis-based candidate gene approach and a hypothesis-generating genome-wide screening approach. In the candidate gene approach, we will sequence selected candidate genes in the IL-12-IFN-? circuit. In the genome-wide screening approach, we will search for and characterize MSMD-causing genes by genome-wide linkage and deep sequencing. By sequencing candidate genes, we have already obtained preliminary evidence of at least three new MSMD-causing defects, with autosomal dominant forms of IFN-?R2, IL-12R?2 and IRF8 deficiency. By genome-wide linkage, we have also obtained preliminary evidence of three novel MSMD-causing genes, with recessive mutations in X-linked CYBB and autosomal TYK2 and JAK2. Our project is innovative, feasible, and supported by strong preliminary evidence. From a basic biological standpoint, this research will provide considerable insight into the mechanisms of immunity to mycobacteria. Elucidation of the molecular genetic basis of MSMD will also shed light on the pathogenesis of mycobacterial disease, making it possible to provide molecular diagnoses for patients and genetic counseling for families. This new information will pave the way for the use of IFN-? for treatment in addition to antibiotics. Finally, the genetic dissection of MSMD will pave the way for the genetic dissection of severe tuberculosis in otherwise healthy children.